The characterization and fractionation of macromolecules, such as DNA and protein, are among the most important diagnostic techniques used in biotechnology today. Heretofore, the most widely used method for fractionation of macromolecules has been a process known as gel electrophoresis.
In a gel electrophoresis process, the macromolecules are forced to migrate through the pores of the gel under the influence of a driving force. Normally, this driving force is a uniform electric field (E), and the velocity of the macromolecules through the gel is dependent on the electric field. The actual direction of macromolecule movement through the electric field (E) will be determined by the charge on the molecule which, depending on the nature of the molecule, may be either positive or negative. It happens that DNA in a fluid medium, such as water, will have a negative charge. It also happens that the speed (i.e. the magnitude of the velocity) at which the macromolecules migrate through the gel is dependent on physical characteristics of the macromolecule. In the case of DNA, it is the length of the various macromolecules which determines their respective speed of migration through the gel. Consequently, after a period of time, a gel electrophoresis process will fractionate the DNA macromolecules according to their length.
Recently, there have been several attempts to perform electrophoresis using micro-sieves rather than gels. Specifically, micro-sieves have been manufactured for this purpose using the well known techniques and processes which were originally developed for the fabrication of integrated circuits (IC) on silicon wafers. In the manufacture of a micro-sieve a pattern of obstacles, rather than circuits, is fabricated on the wafer surface by photolithography and plasma assisted etching. For a micro-sieve, the resultant pattern of obstacles acts much like an obstacle course, or sieve, which impedes the migration of macromolecules across the wafer surface. It happens that the arrangement of the obstacles on the wafer surface, as well as the structural configuration of the obstacles, together determine the operational efficacy of the particular micro-sieve. Several examples can be given.
U.S. Pat. No. 5,427,663 which issued to Austin et al. for an invention entitled "Microlithographic Array for Macromolecule and Cell Fractionation" provides an example wherein a micro-sieve is produced with an array of obstacles which may be of various shapes such as round posts, rectangular bunkers, or v-shaped or cup-shaped structures. According to the disclosure of Austin et al., fractionation of macromolecules and cells is accomplished by impeding the movement of the macromolecules and cells through the micro-sieve. Thus, for the method to work, it is apparent that the size of the obstacles must be comparable to the size of the macromolecules. Macromolecules which are effectively smaller that the spacing between obstacles will be unaffected by the micro-sieve and, therefore, not fractionated.
The importance of being able to fractionate macromolecules which are smaller than the obstacles in a micro-sieve is underscored by the limitations that are imposed by current IC fabrication techniques. At present, the highest resolution for mass produced IC chips using photolithography is approximately 0.25 micron. With electron lithography, this resolution can be improved to approximately 0.1 micron. Electron lithography, however, is a much slower process than photolithography and, in any event, DNA macromolecules are often less than 0.1 micron in length.
To address the obstacle/macromolecule size discrepancy, T. A. J. Duke and R. H. Austin [Phys. Rev. Let. 80 pp. 1552,1998], and D. Ertas [Phys. Rev. Let. 80 pp. 1548, 1998] have independently proposed a method for electrophoresis in which macromolecules are fractionated according to their propensity to diffuse due to the Brownian Motion experienced during their migration through a fluid medium. Specifically, it is known that smaller (i.e. shorter) macromolecules will diffuse more rapidly in a fluid medium than will larger (i.e. longer) macromolecules. Relying on this phenomenon, Duke, Austin, and Ertas have proposed a micro-sieve with obstacles that are asymmetric left and right, relative to the direction of macromolecule migration through the micro-sieve. Due to this asymmetry, the obstacles guide the diffused macromolecules in a lateral direction, and thereby cause the macromolecules to fan out. More specifically, as the macromolecules fan out, they become fractionated according to their propensity to diffuse. For DNA molecules this fractionation results in a separation according to length. Importantly, with a micro-sieve having asymmetric obstacles, macromolecules which are smaller than the spacings between the obstacles can be fractionated. The major disadvantage of this method, however, is that the macromolecules must be injected into the micro-sieve within a very small area (essentially a point source) in order to maintain effective resolution. This results in a very low throughput.
In light of the above, it is an object of the present invention to provide a device with a micro-sieve for fractionating macromolecules in a fluid medium (and its method of use) which is able to fractionate macromolecules that are smaller in size than the spacings between obstacles in the micro-sieve. It is another object of the present invention to provide a device for fractionating macromolecules which has the capacity for a high throughput of macromolecules. Still another object of the present invention is to provide a device for fractionating DNA macromolecules according to the lengths of the DNA macromolecules. Yet another object of the present invention is to provide a device with a micro-sieve for fractionating macromolecules which can sequentially rerun macromolecules through the micro-sieve to improve resolution of the fractionated macromolecules. Another object of the present invention is to provide a device for fractionating macromolecules which is relatively easy to manufacture, is simple to use, and is comparatively cost effective.